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ELUCIDATION OF THE MECHANISM BY WHICH PROTEIN KINASE CK2 PROMOTES CELL SURVIVAL (Spine title: Investigating the role of CK2 in cell survival) (Thesis format: Integrated-Article) by James S. Duncan Graduate Program in Biochemistry A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy School of Graduate & Postdoctoral Studies The University of Western Ontario London, Ontario, Canada © James S. Duncan 2008 Library and Bibliotheque et 1*1 Archives Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-43057-6 Our file Notre reference ISBN: 978-0-494-43057-6 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library permettant a la Bibliotheque et Archives and Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lnternet, prefer, telecommunication or on the Internet, distribuer et vendre des theses partout dans loan, distribute and sell theses le monde, a des fins commerciales ou autres, worldwide, for commercial or non­ sur support microforme, papier, electronique commercial purposes, in microform, et/ou autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in et des droits moraux qui protege cette these. this thesis. Neither the thesis Ni la these ni des extraits substantiels de nor substantial extracts from it celle-ci ne doivent etre imprimes ou autrement may be printed or otherwise reproduits sans son autorisation. reproduced without the author's permission. In compliance with the Canadian Conformement a la loi canadienne Privacy Act some supporting sur la protection de la vie privee, forms may have been removed quelques formulaires secondaires from this thesis. ont ete enleves de cette these. While these forms may be included Bien que ces formulaires in the document page count, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. •*• Canada THE UNIVERSITY OF WESTERN ONTARIO School of Graduate & Postdoctoral Studies CERTIFICATE OF EXAMINATION Supervisor Examiners Dr. David Litchfield Dr. Caroline Schild-Poulter Supervisory Committee Dr. Joseph Torchia Dr. David Haniford Dr. James Koropatnick Dr. David Edgell Dr. Lynn Megeney The thesis by James Stuart Duncan entitled: Elucidation of the Mechanism By Which Protein Kinase CK2 Promotes Cell Survival is accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Date Chair of the Thesis Examination Board ii ABSTRACT Elevated CK2 activity has been associated with the malignant transformation of several tissues, and is associated with aggressive tumor behavior. While the precise roles of CK2 in tumorigenesis remain incompletely understood, mounting evidence suggests a role for CK2 in the protection of cells from apoptosis via the regulation of tumor suppressor and oncogene activity. Consequently, CK2 has emerged as a potential therapeutic target, and strategies to inhibit CK2 have been ongoing in pre-clinical trials. In the present studies, an unbiased evaluation of CK2 inhibitors TBB, TBBz and DMAT was carried out to elucidate the mechanism of action, as well as inhibitor specificity of these compounds. Utilizing a chemo-proteomic approach in conjunction with inhibitor-resistant mutant studies, isoforms CK2a and CK2a' were identified as bona fide targets of TBB, TBBz and DMAT in cells. However, a number of putative off- target inhibitor interactions were identified, including the discovery of a novel TBBz and DMAT (but not TBB) target, the detoxification enzyme Quinone Reductase 2 (QR2). The results described in the present study provide insight into the molecular mechanism of action of the inhibitors, as well as drug specificity, which will assist in the development of more specific next-generation CK2 inhibitors. The convergence of protein kinases and caspase signaling pathways has become increasingly evident, as phosphorylation of a number of caspase substrates within the caspase recognition motif has been shown to prevent caspase cleavage. To investigate the global role of phosphorylation in the regulation of caspase signaling, a novel sequence- based protein identification targeting strategy, (sPITS) was employed. Intriguingly, the constitutively active and oncogenic protein kinase CK2 shares an overlapping requirement with caspases for an acidic consensus sequence, therefore, a comprehensive identification of overlapping CK2/caspase targets was carried out. A number of proteins involved in cell survival were identified, including pro-caspase-3, which was shown to be phosphorylated by CK2 preventing its caspase-dependent activation in cells. Validation of the phosphorylation-dependent protection of pro-caspase-3, as well as identification of numerous candidate CK2/caspase targets in the present studies, support a role for phosphorylation as a global mechanism of regulation of caspase signaling pathways. iii Keywords: Protein kinase CK2, tumorigenesis, phosphorylation, kinase inhibitors, chemo-proteomics, bioinformatics, apoptosis, caspases, mass spectrometry, 2D gel electrophoresis iv CO-AUTHORSHIP STATEMENT The following thesis contains material from previously published manuscripts. David Litchfield is a co-author on all presented papers and was responsible for supervising James S. Duncan during his thesis. For all chapters in this thesis in which a version has been published, James S. Duncan wrote the first draft of the manuscript and David Litchfield played a major role in the editing and revisions of the manuscripts. James Duncan carried out all of the chemo-proteomics experiments described in Chapter 2, prepared all of the figures and wrote the manuscript for publications related to the work of Chapter 2. Laszlo Gyenis and John Lenehan contributed in the preparation and running of a portion of the numerous 2D gels utilized in Chapter 2. Tim Haystead was instrumental in supplying the ATP-sepharose required for the competition assays used in the chemo-proteomics approach, while Lee Graves contributed valuable insight into the proteomic aspects of Chapter 2. Maria Bretner provided a variety of CK2 inhibitors utilized in the chemo-proteomic experiments. James Duncan carried out all of the experiments related to the investigation of phosphorylation as a global mechanism of regulation of caspase signaling, prepared all of the figures and wrote the manuscript for publications related to the work of Chapter 3 and Chapter 4. Greg Gloor was essential in writing and carry out the peptide match program identifying the overlapping CK2/caspase targets from the human proteome, while Shawn Li and Chenggang Wu provided the peptide arrays and fluorescein/biotin labeled peptides. Validation of CSI using mass spectrometry was carried out by James Duncan and Kelly Duncan. Jake Turowec helped purify caspases required for in vitro studies and CSI, as well as performed caspase cleavage assays. v DEDICATION / dedicate this thesis to my loving wife Dr. Kelly Duncan. I would not have made it this far without you. ACKNOWLEDGMENTS First and foremost, I would like to extend my sincerest appreciation to my supervisor, David Litchfield, for all of his guidance and support over the last five years. I also wanted to say thank you for giving me the opportunity to travel to Europe for a conference last summer, it was a very memorable experience. I would like to thank my family: my mom and dad; my sister, Angie and her husband Jeff; my brother-in-law, Brett; and my father- and mother-in-law, Mike and Pat. Their love and support mean the world to me. Sincere thanks to my friends John Lenehan (aka. ICEMAN) and Ryan Mohan who were always their to discuss current topics over some fine beers at the grad club. I'd also like to acknowledge all of my friends in the Litchfield lab over the last five years and the Biochemistry Department, for all of the good times over the years. The biochemistry functions wouldn't have been as fun without you! Many thanks to the following people. My research project would not have been possible without you. • Dr. Greg Gloor for all your helpful discussions regarding the CK2/caspase project • Dr. Shawn Li for critical input into the design and execution of sPITS • Dr. Tim Haystead and Dr. Lee Graves for guidance on experimental design regarding the chemo-proteomics project • Jacob Turowec for all the help with preparing and carry out caspase cleavage assays • Chenggang Wu for preparation of the peptide arrays and fluorescein and biotin labeled peptides • Kystina Jurcic for helping with the protein identification regarding the CK2 inhibitors project vii • Victoria Clarke for all of her help with preparing DNA preps throughout the five years • Chris Ward for contributions in preparing the numerous 2D gels used in the chemo-proteomics studies • CIHR-strategic training program, CIHR Canadian Graduate Scholarship, Ontario Graduate Scholarship, Ontario Graduate Scholarship Science Technology, the Department of Biochemistry and the Faculty of Science for various funding opportunities A very special thanks goes to my wife Kelly for

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